At maturity, about 40% of soybean seed dry weight is protein and 20% extractable oil. These constitute the economically valuable products of the soybean crop. Plant oils for example are the most energy-rich biomass available from plants; they have twice the energy content of carbohydrates. It also requires very little energy to extract plant oils and convert them to fuels. Of the remaining 40% of seed weight, about 10% is soluble carbohydrate. The soluble carbohydrate portion contributes little to the economic value of soybean seeds and the main component of the soluble carbohydrate fraction, raffinosaccharides, are deleterious both to processing and to the food value of soybean meal in monogastric animals (Coon et al., (1988) Proceedings Soybean Utilization Alternatives, Univ. of Minnesota, pp. 203-211).
As the pathways of storage compound biosynthesis in seeds are becoming better understood it is clear that it may be possible to modulate the size of the storage compound pools in plant cells by altering the catalytic activity of specific enzymes in the oil, starch and soluble carbohydrate biosynthetic pathways (Taiz L., et al. Plant Physiology; The Benjamin/Cummings Publishing Company: New York, 1991). For example, studies investigating the over-expression of LPAT and DAGAT showed that the final steps acylating the glycerol backbone exert significant control over flux to lipids in seeds. Seed oil content could also be increased in oil-seed rape by overexpression of a yeast glycerol-3-phosphate dehydrogenase, whereas over-expression of the individual genes involved in de novo fatty acid synthesis in the plastid, such as acetyl-CoA carboxylase and fatty acid synthase, did not substantially alter the amount of lipids accumulated (Vigeolas H., et al, Plant Biotechnology J. 5, 431-441 (2007). A low-seed-oil mutant, wrinkled 1, has been identified in Arabidopsis. The mutation apparently causes a deficiency in the seed-specific regulation of carbohydrate metabolism (Focks, Nicole at al., Plant Physiol. (1998), 118(1), 91-101. There is a continued interest in identifying the genes that encode proteins that can modulate the synthesis of storage compounds, such as oil, protein, starch and soluble carbohydrates, in plants.
Aldolases represent a diverse class of enzymes that differ in their catalytic mechanism and carbonyl donor preference (Wang et al. Biochemistry:44, 9447-9455 (2005)). There are Class I and Class II aldolases. Class II aldolases can be further divided into those that have a preference for dihydroxyacetone phosphate (DHAP) and those that prefer pyruvate as the carbonyl donor. The former represent the best characterized subgroup of Class II aldolases and includes for example fructose-1,6-bisphosphate aldolase, which catalyzes the cleavage of fructose 1,6-bisphosphate into D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, which is the third committed step in glycolysis.
Class II pyruvate-specific aldolases include for example HpaI, a bacterial class II aldolase that catalyzes the reversible cleavage of 2,4-dihydroxy-hept-2-ene-1,7-dioic acid to pyruvate and succinic semialdehyde.
No studies on plant enzymes with similarity to bacterial 2,4-dihydroxy-hept-2-ene-1,7-dioic class II-like aldolase have been conducted and further investigation of the role of this subgroup of proteins in the regulation of storage compounds is therefore merited.
Diacylglycerol acyltransferase (“DGAT”) is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi and mammals. This enzyme is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol (“TAG”). DGAT is associated with membrane and lipid body fractions in plants and fungi, particularly, in oilseeds where it contributes to the storage of carbon used as energy reserves. TAG is believed to be an important chemical for storage of energy in cells. DGAT is known to regulate TAG synthesis. Furthermore, it is known that the DGAT reaction is specific for oil synthesis.
TAG is the primary component of vegetable oil in plants_ It is used by the seed as a stored form of energy to be used during seed germination.
Two different families of DGAT proteins have been identified. The first family of DGAT proteins (“DGAT1”) is related to the acyl-coenzyme A:cholesterol acyltransferase (“ACAT”) and has been described in U.S. Pat. Nos. 6,100,077 and 6,344,548. A second family of DGAT proteins (“DGAT2”) is unrelated to the DGAT1 family and is described in PCT Patent Publication WO 2004/011671 published Feb. 5, 2004. Other references to DGAT genes and their use in plants include PCT Publication Nos. WO2004/011,671, WO1998/055,631, and WO2000/001,713, and US Patent Publication No. 20030115632.
Applicants Assignee's copending published patent application US 2006-0094088 describes genes for DGATs of plants and fungi and their use is in modifying levels of polyunsaturated fatty acids (“PUFAs”) in edible oils.
Applicants' Assignee's published PCT application WO 2005/003322 describes the cloning of phosphatidylcholine diacylglycerol acyltransferase and DGAT2 for altering PUFA and oil content in oleaginous yeast.
Applicants' Assignee's copending published U.S. application Ser. No. 12/470,509 describes DGAT genes from Yarrowia lipolytica combined with plastidic phosphoglucomutase down regulation for increased seed storage lipid production and altered fatty acid profiles in oilseed plants.